Microglia serve as resident immune cells in the brain and are considered key contributors to neuroinflammation, a process that has been implicated in the negative, long-term effects of alcohol on the central nervous system (CNS). The molecular mechanisms underlying activation and related phenotypic transitions of microglia during ethanol exposure are unclear. Based on a recent proteomic analysis of ethanol- treated microglia, we can demonstrate that a significant portion of the ethanol-induced proteome response in microglia could be attributed to changes in the activity of KDM5B, a histone demethylase that catalyzes the removal of tri-methylation on Lys 4 of Histone H3 (H3K4me3). Moreover, we have strong preliminary data that show ethanol induces histone methylation changes both in vitro and in vivo and that experimental modulation of KDM5B activity affects the pro-inflammatory response of microglia. Therefore, we hypothesize that methylation is an important epigenetic modification that influences ethanol-induced activation of microglia resulting in unique functional outcomes that have immediate and possible transgenerational effects on the brain. In order to test our hypothesis, we plan to 1) characterize ethanol-induced changes in the microglial histone methylation code mediated by KDM5B in both primary pure microglia cultures and neuron-microglia co- cultures, 2) determine ethanol dose- and time-dependent role of KDM5B and related histone methylation changes on ethanol-induced microglial activation in vivo and 3) characterize the functional outcome of ethanol- induced epigenetic inheritance of KDM5B-mediated methylation in microglia. The proposed studies incorporate novel approaches such as activity-based protein profiling, methylation-specific quantitative mass spectrometry and ChIP-Seq in order to carry out these aims. This project will be the first ever comprehensive global-scale analysis of methylation and its regulators, which could determine, at least partly, the epigenetic code related to microglial activation phenotype after chronic ethanol exposure in the brain. Characterization of these ethanol- responsive pathways in microglia could also lead to further insight into the development of novel epigenetic therapies for the treatment of CNS dysfunction resulting from alcohol abuse.